Table 1.
Dictyostelium orthologs with a potential role in mechanosensing.
Figure 1.
PKD2 is essential for shear-fluid stress response.
A) Trajectories of WT and pkd2 KO cells migrating randomly (no flow) or subjected to shear flow stress (flow 4 Pa); cells were imaged every 15 sec for a total of 10 min, and the origins were set to 0. Axes indicate distances in µm. The arrow indicates the direction of the flow (from right to left). One representative experiment is shown. B) The directionality of cell migration was assessed by measuring the net displacement on the X axis (Δx). No significant difference between WT and KO cells was seen in control (no flow) condition. Under shear stress, mcln KO cells showed a significant reduction in orientation, while pkd2 KO cells were almost unable to orientate in the direction of the flow. * p<0.05, ** p<0.01, compared to WT values; n = 5. C) The migration speed was calculated as the total distance migrated divided by the time (µm/min). Speed was unchanged upon exposure to a shear stress, and no significant difference was seen between WT and KO cells; n = 5. D) Expression of PKD2 in pkd2 KO cells restores the ability of cells to orientate relative to a shear flow. ** p<0.01, compared to WT values; n = 4. E) Persistence was measured as the net distance between initial and final cell positions divided by the total distance. Here it is shown the ratio between the persistence when cells migrate randomly and when exposed to a shear flow. Only pkd2 KO cells did not show an increased persistence when submitted to a shear stress. ** p<0.01, compared to WT values; n = 5.
Figure 2.
Phylogeny and primary structure of Dictyostelium PKD2.
A) Unrooted maximum likelihood tree of the PKD2 family. Branch lengths are proportional to the number of amino acid substitutions. Numbers at the nodes represent the percentage of bootstrap support (only values >50% are shown). B) Protein alignment of the conserved C-terminal region, spanning TM domains 5 and 6, the pore and coiled-coil regions (boxed). Two other features, only present in the human ortholog, are also indicated: the EF-hand domain and a region essential for endoplasmic reticulum localization. Bold underlined residues are conserved in human (HSP, GenBank accession number NP_000288), C. elegans (CEL, NP_502838), D. melanogaster (DME, NP_609561) and Dictyostelium discoideum (DDI, XP_644933) orthologs. Gaps are denoted by broken lines.
Figure 3.
PKD2 localizes at the plasma membrane.
Confocal images of PKD2-Flag transfected cells, labeled for the FLAG epitope (middle column, green) and cellular markers (left column, red) for late endosomes (p80), plasma membrane (H36), recycling endosomes (p25), contractile vacuole (Rh) and actin (phalloidin). Scale bar: 5 µm.
Figure 4.
PKD2 is essential for calcium-induced lysosome exocytosis.
A) Confocal image of typical exocytic p80 patches in WT and pkd2 KO cells. Scale bar: 5 µm. B) Percentage of cells exhibiting an exocytic patch after transfer to a medium containing 1 mM CaCl2. WT cells showed a rapid and transient increase in fusion events, peaking after 2 minutes, while no induction of lysosome exocytosis was seen for pkd2 KO cells. ** p<0.01, compared to WT values at each time point; n = 5.
Figure 5.
PKD2 is not essential for folate chemotaxis.
A) Cells were deposited at the surface of buffered agar plates 4 mm away to a source of folate (to the left), and allowed to migrate for 5 h. Phase-contrast pictures of one representative experiment after migration are shown. The drawn inner circle represents the position of the cells at time 0. Scale bar: 1 mm. B) Distance travelled by the front of cells between times 0 and 5 h. WT and pkd2 KO cells moved towards folate with similar efficiency; n = 4. C) WT and pkd2 KO cells submerged in phosphate buffer were allowed to move for 90 min towards a micropipette emitting folate. Phase-contrast pictures of one representative experiment are shown at times 0 and 90 min. D) In the experimental setup described in (C), persistence of cell movement was measured as the distance from the initial to the final cell position divided by the total travelled distance. No significant difference was observed between WT and pkd2 KO cells; n = 4. E) The distance of each cell to the micropipette tip was measured at time 0 and 90 min to calculate the overall migration towards the source of folate (Δd). Negative values indicate that cells are moving towards the folate source. No significant difference was observed between WT and pkd2 KO cells; n = 4.